EP0361165B1 - Méthode utilisant des levures immobilisées pour produire de l'éthanol et des boissons alcoolisées - Google Patents

Méthode utilisant des levures immobilisées pour produire de l'éthanol et des boissons alcoolisées Download PDF

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Publication number
EP0361165B1
EP0361165B1 EP89116545A EP89116545A EP0361165B1 EP 0361165 B1 EP0361165 B1 EP 0361165B1 EP 89116545 A EP89116545 A EP 89116545A EP 89116545 A EP89116545 A EP 89116545A EP 0361165 B1 EP0361165 B1 EP 0361165B1
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reactor
carrier
yeast
column
microfibers
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EP0361165A1 (fr
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Heikki Lommi
Juha Ahvenainen
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Danisco Finland Oy
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Cultor Oyj
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00—Fermentation processes for beer
    • C12C11/07—Continuous fermentation
    • C12C11/075—Bioreactors for continuous fermentation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00—Fermentation processes for beer
    • C12C11/02—Pitching yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00—Fermentation processes for beer
    • C12C11/09—Fermentation with immobilised yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C12/00—Processes specially adapted for making special kinds of beer
    • C12C12/04—Beer with low alcohol content
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
    • C12G3/022—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn of botanical genus Oryza, e.g. rice
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/024—Preparation of other alcoholic beverages by fermentation of fruits other than botanical genus Vitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/025—Low-alcohol beverages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00—Fermentation processes for beer
    • C12C11/07—Continuous fermentation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • This invention relates to production of ethanol and more specifically to production of alcoholic beverages using immobilized yeast which is bound to a regenerable carrier material.
  • Yeast immobilization can be carried out in the fermentation reactor itself thus minimizing the risk of contamination during the immobilization.
  • Fermentation is an art that began before recorded history. It was, however, only in 1866 that Pasteur published his works on wine fermentation, analyzed the reasons for spoilage, and prescribed an appropriate treatment for wine making.
  • yeast cell concentration in the fermenting should be increased while on the other hand the ethanol inhibition should be minimized.
  • the yeast cell concentration is 1-2 x 108 cells/ml of must.
  • a higher yeast cell concentration can, for example, be arranged by immobilizing the yeast on a suitable carrier packed in a column.
  • the fermenting must is passed through the column packed with immobilized yeast, the number of yeast cells in contact with the volume of must in the reactor is greatly increased. This greater contact area results in a faster fermentation.
  • the column reactor also reduces the ethanol inhibition since the ethanol containing product is continuously removed from the yeast bed. This reduction is at its greatest when an ideal plug flow situation is achieved.
  • yeast cells can be entrapped in calcium alginate and the resulting immobilized yeast can be used for fast fermentations.
  • Numerous literature references describe this technique as it is applied on laboratory scale. Some efforts, however, have also been made to commercialize this technique.
  • One of the best known is probably that of Kyowa Hakko in Japan, where alginate immobilized yeast is used for feed ethanol production.
  • a major difficulty with alginate entrapment is the manner in which the particles are formed. It must be carried out at the production site where a yeast slurry and a solution of sodium alginate are mixed together. When this mixture is then fed into a calcium-salt solution, the alginate precipitates and at the same time occludes the yeast cells within the precipitated particles.
  • the particles usually are in the form of droplets/beads.
  • a process plant that utilizes alginate entrapped yeast must have specially designed equipment just to produce these beads. Furthermore, there is a potential risk for contaminating the yeast with wild microorganisms. This is especially critical when production of an alcoholic beverage flavor is important (e.g. beer, wine, cider and similar kinds of products). For technical/fuel ethanol production the criteria of contamination is not as important as in the consumables although it does affect the productivity.
  • a second major difficulty for alginate beads used on a commercial scale is in the physical strength of the beads.
  • the beads are soft and easily compressible. Operating large fermentation columns can be a problem and fast downflow process streams are difficult to handle. On the other hand, a typical upflow mode in fermentation greatly wears the beads. Also, to run a reactor downflow under pressure with compressible material is virtually impossible.
  • a third difficulty with entrapment is the diffusion limitations which slow down the accessibility of the substrate in contact with the yeast inside the bead.
  • US-A-4 546 081 concerns a continuous fermentation process for producing ethanol utilizing a fermentation vessel packed with thin film means prepared by mixing an aqueous yeast suspension with photo-crosslinkable resin.
  • an aqueous substrate containing a dissolved yeast-fermentable carbohydrate is contacted with yeast immobilized upon a carrier having anionic exchange properties.
  • the contact can be preferably accomplished by passing the aqueous substrate through a packed bed reactor of the yeast immobilized upon the carrier. However, it may also be accomplished by use of the immobilized yeast in a fludized bed reactor.
  • the yeast-carrier combination is comprised of yeast bound to the surfaces of the porous carrier.
  • the carrier is substantially non-compressible. It is composed of a continuous porous matrix, or alternatively, of dimpled or reticulated, porous particles. The matrix or particles, in turn, are composed of individual microparticles or microfibers.
  • This carrier structure provides a maximal surface area for carrying yeast cells. The resulting high number of yeast cells per unit volume of carrier makes rapid fermentation possible.
  • the particulate or matrix character of the carrier is produced by loosely binding, felting, weaving, gluing or agglomerating (hereinafter binding) the microparticles or microfibers together.
  • the binding is accomplished by establishing chemical, adherent or mechanical links at some of the contact points between the individual microparticles or microfibers. Chemical binding is accomplished by causing a chemical cross-linking reaction at these points.
  • Adherent binding is accomplished by agglomerating or gluing the microfibers or microparticles together through the use of an additional ingredient such as a thermoplastic resin.
  • Mechanical binding is accomplished by entangling or knotting the fibers at the contact points or by joining the particles by meshing their surfaces together.
  • the matrix will comprise a continuous structure throughout the reactor, much like cotton fluff or filter paper packed into a tube. Also then, in their final form, the particles will be discrete and individual.
  • the microfibers or microparticles are composed of any anion exchange substance that can be formed into the desired, rough-surfaced microfibers or microparticles.
  • These substances include native or regenerated cellulose or rayon that is derivatized to provide anion exchange character; synthetic anion exchange resins such as phenolformaldehyde resins and agarose or dextrin based anion exchange resins. Excluded are white birch wood chips.
  • the preferred carrier is a porous, particulate anion exchange resin derived from cellulose or rayon that has been chemically modified to provide anion exchange character.
  • Especially preferred embodiments include microfibers or microparticles of diethylaminoethylene substituted cellulose, adherently bound by agglomeration with polystyrene.
  • the aqueous substrate used as a starting material is composed at least of water and a fermentable carbohydrate such as hydrolyzed starch, sucrose, glucose, fructose, maltose or maltotriose (lactose and xylose).
  • a fermentable carbohydrate such as hydrolyzed starch, sucrose, glucose, fructose, maltose or maltotriose (lactose and xylose).
  • concentration of sugar will be sufficient to permit continuous production of alcohol but will not be so high that the fermentive activity of the yeast is completely inhibited.
  • the process parameter involving carbon dioxide produced by the fermentation it is important to control the process parameter involving carbon dioxide produced by the fermentation.
  • the carbon dioxide can be maintained in the fluid product stream or removed.
  • a series of columns is employed, the interconnections of which are adapted for removal of gas from the column outlet stream. If the column interconnection taps are all employed, a product without carbonation will be produced.
  • the column pressure cannot be so high as to substantially diminish the yeast fermentation. Facile routineering can establish this limit, which, in general, will be at least about 14 bar.
  • process parameters can be varied to affect the output of ethanol and taste of the product.
  • These parameters include column temperature, aqueous substrate feed rate, column residence time, direction of substrate flow in the column (with or against gravity), periodic reversal of flow direction, yeast strain, yeast concentration and yeast nutrients in the aqueous substrate. Suitable ranges for these parameters include a temperature of -2 to 40°C, feed rate 0.01-10 reactor bed volume (BV)/hour, a residence time of 0.1-100 hours and yeast concentration of 109 to 1012 yeast cells per litre of carrier. Yeasts such as Saccharomyces or Candida can be used. Generally, these parameters will be adjusted to produce an ethanol concentration of from 0.05 to 15 percent.
  • a slurry of the particulate carrier is first placed in a column and the carrier allowed to settle into a packed column.
  • the carrier is sterilized by a method known per se, such as washing with hot caustic.
  • the column is then flowed with a yeast broth so that the yeast becomes attached to and immobilized on the carrier particles.
  • the column of yeast is used as described above.
  • an ethanolic product that is consumable is produced.
  • This product can be wine, sake, an alcoholic fruit, berry or vegetable drink, carbonated versions thereof, beer, or low alcohol versions of the foregoing products.
  • the aqueous substrate used to produce the consumable product is derived from a source such as a fruit, berry or vegetable juice or extract, a wort, hydrolyzed plant material or an aqueous syrup containing a fermentable sugar derived from a natural or synthetic source.
  • the juice will be a liquid pressed from a fruit, berry or vegetable.
  • the extract will be a liquid produced by combining the fruit, berry or vegetable with water and processing by mashing, cooking, pressing, mixing and the like.
  • the hydrolyzed plant material will be material that is derived from cellulose, hemicellulose and/or starch through a technique such as acid, enzyme or auto-hydrolysis.
  • Another method of the invention is directed to production of low alcohol beverages such as those containing for example less than 0.2 percent by volume alcohol.
  • the steps practiced and the yeast-carrier material are the same as those of the primary production method.
  • the sugar content and feed rate of aqueous substrate are modified to provide the "low alcohol" production.
  • the feed rate is increased to an extent that decreases the alcohol concentration to the value desired.
  • the sugar concentration is also appropriately adjusted so that the beverage is not overly sweet yet enough sugar is present for fermentation to alcohol.
  • the invention is further directed to the combination of the carrier and yeast in any form.
  • Preferred forms include that made in situ , as described above, and a dried combination.
  • the dried combination in aseptic condition can be packaged, transported to a fermentation plant and reconstituted for use by immersion in aqueous nutrient. Alternatively, the dried form can be packaged for home use.
  • the Figure is a flow scheme for preparing the immobilized yeast columns.
  • the Figure shows a hydration vessel 1, an immobilized yeast reactor 2, a sterilization liquid vessel 3, a neutralization acid vessel 4, a yeast slurry vessel 5, a substrate vessel 6, a carbon dioxide separation port 7, a receiving vessel 8 and a pressure releasing valve 9.
  • the present invention is based upon a yeast-carrier system, described above, that is non-compressible and sterilizable.
  • a specially preferred example of a carrier for use in producing the yeast-carrier system is particulate DEAE cellulose (weak anion exchanger) agglomerated with polystyrene. This carrier is commercially available and is well-known for immobilizing enzymes typically isomerase (see U.S. Patent No. 4,355,117 for details regarding this carrier).
  • the dry carrier is hydrated in a hydration vessel 1 and pumped into a column reactor 2.
  • the preferred DEAE cellulose carrier is stable in acid and alkaline media and can withstand a temperature up to 100°C. It can thus be easily sterilized in the column, for example, with hot caustic alkali. Other resins can appropriately be sterilized in a similar way.
  • Yeast immobilization onto the carrier is accomplished after sterilization of the carrier with hot dilute caustic alkali 3.
  • the carrier bed 2 is then rinsed with water and neutralized by pumping a suitable dilute acid 4 through the carrier bed; finally the carrier bed is rinsed with sterile water.
  • the immobilization is carrier out by pumping a cultured, active yeast slurry 5 into the reactor 2.
  • the carrier then adsorbs the yeast.
  • the yeast cells attach to the carrier surfaces as a result of the optimal surface shapes of the carrier.
  • the yeast cells are grown on the carrier to provide a density of between about 109 - 1012 cells per litre of carrier, with a density of about 109 cells being particularly preferred.
  • the density must be sufficient to provide enough enzymatic activity to substantially convert the sugars in the aqueous substrate to ethanol.
  • the substrate from vessel 6 is pumped through the reactor either through the bottom or top inlet.
  • Slow flow rate (about 1BV/day) means long contact time between the carrier and the substrate and thus high alcohol levels. Faster flow again lowers the alcohol level.
  • the reactor can be run under atmospheric pressure feeding from the bottom inlet the carbon dioxide being separated from the system freely through a separation port 7.
  • the product is collected to a receiving vessel 8 from where it is further processed through filtration etc. before final bottling.
  • the substrate 6 When the system is operated under pressure, the substrate 6 is fed from the top inlet in order to keep the carrier bed in packed form and thus reach a plug flow.
  • the fermentation rate is controlled in such a manner (by flow rate) that the applied pressure is high enough to maintain the formed carbon dioxide dissolved.
  • the pressure is released through valve 9 on the receiving vessel unless the final product is intended to contain carbon dioxide.
  • the carrier can be regenerated by feeding a hot caustic alkali through the carrier bed until the material colour is uniformly bright. Then the carrier is rinsed with water until a pH of about 10 is achieved and neutralized by pumping a suitable dilute acid through the carrier bed. Finally, the carrier is rinsed with sterile water.
  • the carrier is important in terms of providing an adequate environment for yeast growth and contact with the aqueous substrate.
  • the non-compressible anion exchange carrier such as agglomerated DEAE-cellulose, has several beneficial qualities compared with soft, gel-like carriers, such as alginate beads. These qualities include fewer mass transfer problems, easier immobilization, faster start-up, easier scale-up, much improved regenerability and a long life-time of the carrier.
  • Electron micrographs show that in the alginate beads, the yeast grows in colonies, some of which grow through the alginate layer to the bead surface. The yeast colonies presumably act as the "active sites" in the alginate beads.
  • the microphotographs show that the DEAE-cellulose granules are porous, reticulated matrices of microfibers. This structure permits the aqueous substrate to reach yeast cells growing inside the granules. Thus, the yeast grows rather loosely and separately in the internal and external pockets of the microfibers and these individual cells act as "active fermentation sites” in the column. Accordingly, more yeast cells per unit carrier surface area are available for fermentation when the carrier of the invention is used.
  • the mechanism of the yeast cell immobilization onto the surface of the carrier offers many benefits.
  • the yeast cells are substantially all on the surface of the carrier.
  • the cells act as if they were freely suspended in a solution.
  • the carrier properties according to the invention also permit easy start-up and regeneration because the yeast cell immobilization can take place in situ within the column. This procedure lowers the risk of contamination and improves the conditions of the system as a whole.
  • the column can be easily regenerated which is an important economic advantage. Regeneration is sometimes expedient because of chemical impurities or contamination from the aqueous, microbial contamination or from mutations of the immobilization yeast itself.
  • the spent column can be washed and sterilized with a hot caustic solution or with another sterilizing medium such as an organic agent in water and the like. After washing and neuralization, the carrier is ready for repitching and reimmobilization.
  • pilot scale set-ups such a column has been utilized for at least 13 weeks without need for regeneration.
  • laboratory scale a similar column has been utilized for about 30 weeks.
  • the entire reactor system can be sterilized with the above-mentioned treatment.
  • an aseptically cultured yeast is then pumped or eluted through the carrier bed, the risk of contaminating the yeast/carrier with wild strains or bacteria will be minimal.
  • the yeast is attached to the column (a suitable charge is about 109 to 1012 yeast cells/litre carrier)
  • the column can be further conditioned by slowly pumping a nutrient solution such as an aqueous medium of ammonium phosphate and sugar through the reactor for approximately one day. This process causes the yeast to flourish to a maximum density.
  • one of the preferred carrier embodiments is non-compressible (non-swelling). This property allows much variation as to how the fermentation can be conducted.
  • the reactor/continuous fermenter may be operated as follows.
  • the substrate is fed in from the bottom of the reactor and the CO2, which is formed during the fermentation, is freely allowed to evolve from the carrier bed.
  • CO2 bubbles always disturb the bed
  • backmixing causes ethanol inhibition and thus lower productivity.
  • the non-compressible carrier system of the present invention can also be operated under increased pressure in order to maintain the carbon dioxide in a dissolved state.
  • the column can then also be run in a downflow condition and in this packed bed system, an ideal plug flow is reached.
  • the flow of the aqueous substrate through the reactor is periodically reversed.
  • pressurized columns may also be run in series to avoid high pressure.
  • the series arrangement is especially useful if a large concentration of alcohol in the final product is desired.
  • This method enables the operation in a downflow mode without the harmful release of carbon dioxide which could disrupt the packed character of the column, i.e. cause channelling. Between the columns, carbon dioxide is separated from the stream.
  • the reactor size can also be kept smaller as no extra fluidization and carbon dioxide-separation space is needed.
  • any fermentable fluid substrate can be used as feed material for production of the ethanolic product provided that any particulate impurities are filtered out before feeding into the immobilized yeast column.
  • Examples of typical substrates would be:
  • the flow rate of the aqueous substrate through the column is an important parameter because the ethanol production capacity is dependent on the flow rate. It has also been shown that the flow rate determines the number of yest cells which remain bound in the carrier. With high flow rates, the yeast cell outflow is increased; consequently ethanol production may also be dependent on the cell number bound in the immobilized system, and not only on the throughput rate of the substrate. If the flow rate is too high, the ethanol production is incomplete and/or inadequate; by controlling the flow rate, the ethanol concentration can be controlled, typically by seeking a concentration which is from 0.05 percent to 15 percent by volume relative to the total volume of the aqueous substrate. By adjusting the flow rate, the fermenter can obtain an ethanol concentration within this range.
  • the amount of yeast cells which remain bound to the carrier material appears to be relatively stable at low flow rates, suggesting that yeast growth and leaching of the cells are balanced. With higher flow rates leaching increases but the cell number returns back to the normal level again when the flow rate is decreased.
  • the flow rate should be high enough to enable the leaching of dead cells in order to avoid autolysis.
  • the yeast is maintained viable by fermentable sugars in the aqueous substrate.
  • the immobilized yeast column reactor can be pressurized as discussed above.
  • the operation pressure should be high enough to keep the carbon dioxide soluble in the column and to avoid the possible channelling of the CO2 bubbles through the immobilized yeast column. Typical measure is 14 bar.
  • a suitable temperature is that used in traditional batch fermentation processes. It will range from 0 to 40°C, preferably 10 to 35°C. This temperature can be maintained either by warming the columns with heating jackets or maintaining the columns in an environmentally controlled room, or by cooling. Of course, the yeast also develops a certain degree of heat that also can be used to advantage.
  • the pressure is chosen according to the operation temperature.
  • the eluted ethanolic product is cooled down to the storage temperature and collected into a buffer tank for the conventional post-fermentation treatment, e.g. filtering, pasteurization and packaging.
  • the ethanol production system described above is a faster and more convenient system than traditional fermentation processes and produces a product with acceptable taste equivalent to the taste resulting from conventional fermentation processes.
  • the time needed for fermentation is reduced from weeks to a matter of hours by the present invention which, along with the continuous nature of the process, provides a vast potential for time and money savings in commercial alcohol or alcoholic beverages production.
  • the carrier was filled, sterilized and yeast immobilized thereon according to the following procedure for handling GDC:
  • the hydration vessel (1) was first filled half with water.
  • the mixer was started and dry carrier (GDC) was transferred to the vessel (1).
  • GDC dry carrier
  • the immobilized yeast reactor (2) was filled half with water and the carrier water slurry from the hydration vessel (1) was transferred to the reactor (2).
  • the bottom valve on the reactor was adjusted so that inlet and outlet flows from the reactor were about the same.
  • the carrier in the reactor was then sterilized with hot dilute caustic alkali (3) by pumping it through the reactor (2).
  • the carrier bed was then rinsed with water and neutralized by pumping a suitable dilute acid (4) through the carrier bed in the reactor (2) and finally the carrier bed was rinsed with sterile water.
  • a yeast slurry was made up in the vessel (5).
  • the yeast slurry was then pumped through the carrier bed in about 1-4 hours whereby the yeast was adsorbed onto the carrier.
  • the yeast was now immobilized onto the carrier.
  • the reactor (2) was basically ready for fermentation.
  • the yeast was cultured as it is done for batch fermentations and the carrier beds of about 500 ml each were prepared with an amount of yeast cells as described in Example 1.
  • the carrier had a particle size of 0.315 to 0.840 mm.
  • the substrate feed was started with a flow rate of 1 bed volume/day.
  • the substrate was pasteurized apple juice where 1 g/l of ammonium phosphate was added as yeast nutrient.
  • the total sugar concentration was adjusted to 220 g/l by adding sugar to the apple juice.
  • the fermentation started slowly and after 3 weeks the ethanol level was about 4.5%.
  • Example 2 was repeated using a column loaded with a carrier of the particle size 0.470 - 0.840 mm. After 50 days operation the flow rate was reduced to about 0.8 bed volume/day which immediately increased the ethanol level during the following week from 5% to 7-8%.
  • Example 3 was repeated using a column which was wider and shorter (diameter 75 mm and height 152 mm).
  • the yeast immobilization was carried out with substantially higher yeast cell concentration: for 500 ml of GDC, 600 ml of yeast slurry was used which contained 150 million cells/ml.
  • the leakage through the column was 5 million cells/ml so the total immobilized amount was 9 x 1010 cells. This is equal to 2 x 109 cells/ml GDC or 5 x 109 cells/g GDC.
  • Example 4 was repeated using an 8 litres reactor.
  • the reactor was constructed so that in the outlet line there was also a screen plate to hold back any GDC particles.
  • the system stabilized into a 10 % ethanol level within a week.
  • a portion of the product from the 8 litres reactor was further fed to a second 500 ml column to increase the level of ethanol.
  • the column preparation and yeast immobilization was carried out following the procedure described in Example 1.
  • a 500 ml carrier bed was packed in a glass column which was wider in the upper end in order to ease the carbon dioxide separation.
  • the wort was fed into the bottom of the fermentation column.
  • the yeast cell concentration was adjusted to 109 cells/g GDC.
  • wort Traditional wort for lager beer brewing was used as a feed material.
  • the wort was produced from 18 kg of barley malt (pilsner type) which gave a final volume of 100 l of finished wort (12.0°P).
  • the mixture of water and malt grist was mashed in one vessel by programmed infusion method with rests at temperature 48°C for 15 minutes, 63°C for 30 minutes, 72°C for 20 minutes and 78°C at the end of mashing.
  • the wort was clarified in a lauter tun and rinsed twice with 78°C water.
  • the wort was boiled in a wort kettle for about 90 minutes. Hop pellets were added at the beginning of boiling (total amount of alpha acids was about 10 g). Any precipitation formed during boiling was separated in a whirlpool.
  • the clarified wort was cooled in a plate heat exchanger from 100°C to 10°C.
  • composition of the wort was as follows: Original extract 12.0 °Plato Colour 10.0 °EBC Bitterness 25 EBU pH 5.3 Apparent attenuation rate 85%
  • composition of the wort can vary as follows: Original extract 6-18 °Plato pH 4.5-5.5 Apparent attenuation rate 65-100%
  • the rice was first liqufied by steaming and hydrolyzing with alphaamylase and amyloglucosidase.
  • the resulting hydrolysate was filtered in order to remove all mechanical impurities and unhydrolyzed residues.
  • the substrate was finally diluted so that the glucose concentration was 23% when it was fed into the column.
  • the column had a carrier colume of about 500 ml.
  • the column diameter was 70 mm and diameter to height ratio of the carrier about 1:2. Comparison was carried out with Ca-alginate entrapped yeast. The feed rates, parameters and results for both fermentations are given in the following table.
  • the fermentation rate was controlled by regulating the feed rate and the required flavour and alcohol content in the product was achieved.
  • the column preparation and yeast immobilization was carried out following the procedure described in Example 1.
  • the yeast cell concentration was adapted to provide 109 cells/g GDC.
  • the aqueous substrate for fermentation was formulated as follows: Crystal (white) sugar 500 g Honey 125 g Soft brown sugar 625 g Juice of 2 lemons Water 10 l
  • the column has a carrier volume of 500 ml.
  • the substrate was fed into the bottom.
  • the feed rate at the beginning was 1 bed volume per 5-10 hours. After stabilization of fermentation the feed rate was regulated to 1 bed volume per 1 - 5 hours depending on the ethanol content and sweetness desired in the product.
  • the ethanol content was between 0.2 and 2.0% w/w in the beverage.
  • small amounts of ammonium phosphate (10-100 ppm) were added as a nutrient.
  • the production was carried out at room temperature with the parameters described above.
  • the immobilized yeast reactor can also be operated under pressure at lower temperature. Then the carbon dioxide formed is dissolved and final addition of carbon dioxide can be avoided.
  • raw materials can also be used as substrates. All contain fermentable sugars, e.g. malt, fruits and berries.
  • the flavour of the final product will depend on raw material, sugar concentration and fermentation rate.
  • the resins were: Granulated DEAE cellulose having the trademark SPEZYME GDC 220 and a synthetic anion exchange resin having the trademark DUOLITE A 568.
  • the immobilizations were performed according to the following procedure: The yeast was incubated for 48 hours in a malt extract broth at 30°C. The resin was sterilized by washing with 1 M sodium hydroxide, buffered to pH 5.0 - 5.1 and washed with sterile water. A 10 g dry weight sample of the resin was flushed in a 20 mm i.d. glass column equipped with a glass sinter bottom plate. 100 ml of the yeast suspension was passed by gravity through the column at an approximate rate of 3 bed volumes/hour, after which the column was washed with 100 ml of sterile water.
  • the cell concentration of the yeast suspension before and after the immobilization was determined by an agar plate count. From the difference, the number of immobilized cells was calculated.
  • the results as immobilized cells per gram of resin were as follows: Resin Yeast Offered Immobilized SPEZYME® A-75050 2.3 x 108 2.1 x 108 DUOLITE® A-75050 2.3 x 108 2.2 x 108
  • the DEAE cellulose column used in the experiments was regenerated by feeding a hot (about 60°C) caustic soda solution (2% sodium hydroxide) through the column until the material colour was uniformly bright.
  • the column was rinsed with sterile water until the pH of the leaving solution was about 10, and neutralized with sodium pyrosulphite to a pH of about 7.
  • the column was rinsed with sterile water and filled with wort, pitched with yeast cells (about 1010 cells/litre of carrier), the cells were grown in the presence of aerated wort for 24 hours and were then ready for fermentation of the substrate.

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  • Life Sciences & Earth Sciences (AREA)
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Claims (14)

  1. Procédé de production d'un produit éthanolique, caractérisé en ce que dans l'étape de production principale, on fait passer un substrat aqueux contenant un sucre fermentescible dans un réacteur contenant des cellules de levure immobilisées sur les surfaces d'un support sensiblement non comprimable, composé par une matrice poreuse continue ou par des particules poreuses creuses ou réticulées, ladite matrice ou lesdites particules présentant une structure formée de plusieurs microparticules ou microfibres associées de façon lâche, qui sont liées ensemble chimiquement, par adhérence ou mécaniquement au moins en certains points de contact entre les microparticules ou microfibres individuelles, lesdites microparticules ou microfibres étant composées d'une résine échangeuse d'anions, à l'exclusion des copeaux de bouleau blanc.
  2. Procédé selon la revendication 1, caractérisé par :
       le chargement d'un réacteur par un mélange aqueux d'un support sensiblement non comprimable tel que défini dans la revendication 1 ;
       la stérilisation éventuelle du réacteur chargé ;
       l'adsorption d'un bouillon de levure sur le support ;
       l'introduction d'un substrat aqueux contenant un sucre fermentescible dans le réacteur contenant la levure immobilisée ; et
       la récupération du produit éthanolique.
  3. Procédé selon les revendications 1 et 2, caractérisé en ce que les microparticules ou microfibres composées par une résine échangeuse d'anions sont choisies dans le groupe constitué par une cellulose native ou régénérée transformée en dérivé pour lui donner un caractère d'échangeur d'anions.
  4. Procédé selon les revendications 1 à 3, caractérisé en ce que les microparticules ou microfibres sont liées ensemble par des liaisons par adhérence.
  5. Procédé selon les revendications 1 à 3, caractérisé en ce que le support contient des particules formées de microfibres agglomérées avec du polystyrène et en ce que la résine échangeuse d'anions est la cellulose à substituants diéthylaminoéthylène.
  6. Procédé selon les revendications 1 à 5, caractérisé par :
       la régénération de la capacité de fermentation d'un réacteur par élimination de la levure immobilisée du support garnissant le réacteur, et
       le passage d'un nouveau bouillon de levure dans la colonne pour provoquer l'immobilisation de nouvelles cellules de levure sur le support.
  7. Procédé selon la revendication 6, caractérisé par :
       l'élimination des cellules immobilisées par passage d'un alcali caustique chaud dans la colonne,
       le rinçage de la colonne par de l'eau,
       la neutralisation par passage d'un acide dilué dans la colonne et
       le rinçage par de l'eau et
       le passage d'un nouveau bouillon de levure dans la colonne.
  8. Procédé selon les revendications 1 à 7, caractérisé par le passage du substrat aqueux dans plusieurs réacteurs placés en série et possédant des moyens d'élimination de gaz du liquide quittant chaque réacteur.
  9. Procédé selon les revendications 1 à 8, caractérisé par le passage du substrat aqueux dans le réacteur sous une pression suffisante pour maintenir à l'état dissous une partie importante du dioxyde de carbone produit, produisant ainsi un produit éthanolique gazeux.
  10. Procédé selon les revendications 1 à 9, caractérisé en ce que l'écoulement du substrat aqueux dans le réacteur se trouve dans une direction s'opposant à la force de gravité.
  11. Procédé selon les revendications 1 à 9, caractérisé par l'inversion périodique de l'écoulement du substrat aqueux dans le réacteur.
  12. Dispositif de production d'un produit éthanolique contenant :
    a) un réacteur (2) contenant des cellules de levure immobilisées sur les surfaces d'un support sensiblement non comprimable, composé par une matrice poreuse continue ou par des particules poreuses creuses ou réticulées, ladite matrice ou lesdites particules présentant une structure formée de plusieurs microparticules ou microfibres associées de façon lâche, qui sont liées ensemble chimiquement, par adhérence ou mécaniquement au moins en certains points de contact entre les microparticules ou microfibres individuelles, lesdites microparticules ou microfibres étant constituées par une résine échangeuse d'anions, à l'exclusion des copeaux de bouleau blanc, ce réacteur (2) étant associé avec :
    b) un récipient (1) d'hydratation du support sec avant son pompage dans le réacteur (2),
    c) un récipient (3) contenant un liquide de stérilisation destiné à stériliser le support contenu dans le réacteur (2),
    d) un récipient (4) contenant un acide de neutralisation destiné à neutraliser le support après stérilisation,
    e) un récipient (5) contenant la suspension épaisse de levure à pomper dans le réacteur (2) et à lier sur la surface du support,
    f) un récipient (6) contenant le substrat à traiter par le réacteur (2),
    g) des orifices de séparation (7) du dioxyde de carbone,
    h) un récipient (8) de réception du produit final après le traitement dans le réacteur (2) et
    i) des valves de détente de la pression (9).
  13. Dispositif selon la revendication 12, dans lequel le réacteur (2) comprend plusieurs colonnes sous pression en série.
  14. Dispositif selon la revendication 13, dans lequel on place entre les colonnes des moyens de séparation du dioxyde de carbone du courant.
EP89116545A 1988-09-27 1989-09-07 Méthode utilisant des levures immobilisées pour produire de l'éthanol et des boissons alcoolisées Expired - Lifetime EP0361165B1 (fr)

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US07/249,898 US5079011A (en) 1988-09-27 1988-09-27 Method using immobilized yeast to produce ethanol and alcoholic beverages
US249898 1988-09-27

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EP0361165B1 true EP0361165B1 (fr) 1994-11-30

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JP (1) JP2695942B2 (fr)
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AT (1) ATE114721T1 (fr)
AU (1) AU629849B2 (fr)
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DE (1) DE68919620T2 (fr)
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DE4003404A1 (de) * 1990-02-05 1991-08-08 Moselland Eg Winzergenossensch Gaerprodukt mit vermindertem ethanolgehalt
US5612072A (en) * 1990-10-23 1997-03-18 Cultor Ltd. Process for the production of non-alcoholic or low alcohol malt beverage
DE4239612A1 (de) * 1992-11-25 1994-05-26 Cultor Oy Bioreaktor mit immobilisierten, Milchsäure-produzierenden Bakterien und dessen Verwendung in Fermentationsverfahren
GB9227134D0 (en) * 1992-12-31 1993-02-24 Fuller Jess P Cell support structure
CA2133789A1 (fr) * 1994-10-06 1996-04-07 Ronald James Neufeld Production de perles de carraghenane a cellules immobilisees; methode de brassage utilisant des cellules de levure immobilisees de perles de carraghenane
CA2188971A1 (fr) 1995-10-27 1997-04-28 Beatriz Sanchez Boisson de malt possedant un gout stabilise; methodes de preparation
KR100440723B1 (ko) * 1996-12-30 2004-10-02 오비맥주 주식회사 고정화효모를이용한맥주의제조방법
US6013491A (en) * 1997-08-06 2000-01-11 Martinez; Leticia Fibrous cellulose support containing adhered yeast for converting sucrose to glucose and fructose
DE19848623A1 (de) * 1998-10-21 2000-04-27 Cultor Corp Preiswerte Immobilisierungsmatrizen aus natürlichen Materialien
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FI894205A0 (fi) 1989-09-06
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ATE114721T1 (de) 1994-12-15
US5079011A (en) 1992-01-07
DK472989A (da) 1990-03-28
AU629849B2 (en) 1992-10-15
FI87658B (fi) 1992-10-30
LV10501A (lv) 1995-02-20
FI87658C (fi) 1993-02-10
RU2060277C1 (ru) 1996-05-20
JPH02276583A (ja) 1990-11-13
FI894205A (fi) 1990-03-28
EP0361165A1 (fr) 1990-04-04
JP2695942B2 (ja) 1998-01-14
ES2067510T3 (es) 1995-04-01
DE68919620T2 (de) 1995-05-11
NZ230564A (en) 1991-07-26
DK174923B1 (da) 2004-02-23
DK472989D0 (da) 1989-09-26
KR900004929A (ko) 1990-04-13
LV10501B (en) 1995-10-20
LT3668B (en) 1996-01-25
KR0153762B1 (en) 1998-10-01
AU4104289A (en) 1990-04-05

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